Generation of Reactive Oxygen

Species by Mitochondria
Dr. Derick Han
Dr. Enrique Cadenas

Generation of Reactive Oxygen

Species by Mitochondria

Dr. Derick Han* and Dr. Enrique Cadenas#

* USC Research Center for Liver Diseases,

Keck School of Medicine
# USC School of Pharmacy
University of Southern California,
Los Angeles, California 90033 – USA

Introduction

Mitochondria are the major

producers of ATP in cells

Mitochondria are also the major

sources of reactive oxygen species
(e.g., superoxide anion (O2.–)
and H 2O2) in cells

Reactive oxygen species generation

by mitochondria is linked
with ATP production
by mitochondria (electron transport
along the respiratory chain)

Oxygen chemistry

2s 2p

O O
e-

ATP
H2O

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 Generation of Reactive Oxygen
Species by Mitochondria
Dr. Derick Han
Dr. Enrique Cadenas
Reactive oxygen species chemistry
Lewis dot diagram Reactiv e oxygen species are generated
through electron transfer reactions with oxygen

o2 o2 -
O O Molecular oxygen has two unpaired
electrons; Hence, it is a radical +1e-

Molecular O O O O
oxygen +1e-
Molecular Superoxide
oxygen anion

O O Superoxide anion has one unpaired

electron; Hence, it is a radical

Superoxide O O O O
anion +1e-
Superoxide (●) Hydrogen
anion peroxide

Hydrogen peroxide has no unpaired

H O O H electrons; Hence, it is not a radical
Fe2+
Hydrogen H O O H H O + O H
peroxide +1e-
Hydrogen (●) Hydroxyl Hydroxyl
Annu. Rev. Biochem., 1989, 58: 79-110 peroxide radical
4
anion

Reactive oxygen species

mediated cellular damage
Nucleobase oxidation by hydroxyl radical
Superoxide damage O O
to aconitase, a citric acid N + OH N
HN HN
cycle protein OH
H2 N N N H2 N N N
R R
S–Cys
S–Cys deoxy-
desoxy- 8-hydroxy-
8-hydroxy-
guanosine
guanosine deoxyguanosine
desoxyguanosi ne
Protein crosslink by hydroxyl radical
O2.– S–Cys

[Fe3 S 4 ]+

S–Cys .OH

S–Cys
S–Cys

Tyr
S–Cys
[Fe4 S 4 ]2+
5

Reactive oxygen species in disease

Increasing evidence suggests that the generation of these reactive oxygen species plays
an important role in the pathophysiology of diseases such as:
ischemia reperfusion injury, neurodegenerative disorders and aging
Other clinical conditions in which the involvement of oxygen radicals has been suggested are:
• Skin
- Porphyria • Red blood cells
- Hemolytic anemia
- Solar radiation
- Protoporphyrin photooxidation
• Eye
- Lead poisoning
- Cataract
- Phenylhydrazine toxicity
- Retrolental fibroplasia
- Primaquine and related drugs
• Cardiovascular system
• Lung
- Keshan disease (selenium deficiency)
- Emphysema
- Atherosclerosis
- Bleomycin toxicity
- Adriamycin cardiotoxicity)
• Brain - Paraquat toxicity
- Parkinson’s disease - Asbestos carcinogenicity
- Alzheimer’s disease
- Multiple sclerosis - Rheumatoid arthritis
- Neurotoxins - Autoimmune diseases

• Inflammatory - immune injury

• Ischemia-reperfusion states
- Myocardial infarction / stroke
- Organ transplantation
- Frostbite 6

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 Generation of Reactive Oxygen
Species by Mitochondria
Dr. Derick Han
Dr. Enrique Cadenas
Antioxidant defenses
Catalase: this enzyme is located in the peroxisomes and catalyses the following : H2O 2 + H2O 2 → 2H2O + O 2

Glutathione peroxidase: the enzyme occurs in cytosol and the mitochondrial matrix and it requires glutathione,
a tripeptide present in high concentrations in most mammalian cells; H2O 2 + 2GSH → H2O + GSSG

Glutathione peroxidase

Catalase

O2 O2.- H2O2 HO. H2O

Superoxide dismutase Vitamin E

Vitamin C
Ubiquinol
Spontaneous nonenzymatic dismutation Uric acid
O 2.- + O 2.- + 2H+ H202 + O 2 105 M-1S-1 Glutathione

O 2.- + O 2.- + 2H+ H202 + O 2 Enzymatic dismutation

SOD 109 M-1S-1

Superoxide dismutases: (abbrev iated SOD) catalyze the rapid dismutation

of superoxide radical to hydrogen peroxide and oxygen
7

Overview of mitochondrial reactive

oxygen species generation

I. Mitochondria structure and topology

II. Bioenergetics

III. Reactive oxygen species generation by mitochondria

a) Sites and mechanism
b) Topology

IV. Methods to measure reactive oxygen species

V. Regulation of mitochondrial
reactive oxygen species generation

I. Mitochondria structure and topology

Inter membrane space
Inner membrane contains cytochrome c as well as many
Contains respiratory complexes apoptotic factors (e.g., AIF, SMAC)
for oxidative phosphorylation, ATP synthase,
ATP/ADP translocator Cristae
as well as many transporting proteins; Increases surface area
The inner membrane is impermeable to ions of the inner membrane
(especially H+) for oxidative phosphorylation

Matrix Outer membrane

Contains pyruvate dehydrogenase,
citric acid cycle enzymes, fatty acid
b-oxidation enzymes, some urea cycle
and metabolic enzymes
Due to voltage dependent anion channels
(VDAC) and other channels, the outer
membrane is fairly permeable to ions
and small molecules
9

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 Generation of Reactive Oxygen
Species by Mitochondria
Dr. Derick Han
Dr. Enrique Cadenas

Mitochondria: a major cellular source

of oxygen radicals

Sources of O2.– and H2O2

• Outer mitochondrial membrane

– Monoamine oxidase (MAO)

• Inner mitochondrial membran e

H2O2
– Respirator y chain

O2.–

O2
MAO

O2 H2O2

10

I. Mitochondria structure and topology –

respiratory complexes

Respiratory complexes
Succinate
dehydrogen ase
Cytochrome
oxidase
II

I I UQ III IV V

cyt c
NADH-ubiquinol ATP
Ubiquinol-
oxidoreductase synthase
cytochrom e
oxidoreductase

11

II. Mitochondria bioenergetics

Matrix

Succinate
H+
NADH
H+ e+ H+ ADP ATP
H+
e+ O2 2 H2O
II
4e+
I I UQ III IV V

Inner cyt c
membrane
H+ H+ H+ H+ H+
H+ H+ H+

Outer VDAC
membrane 12

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 Generation of Reactive Oxygen
Species by Mitochondria
Dr. Derick Han
Dr. Enrique Cadenas
III. Reactive oxygen species generation
by mitochondria – overview
Succinate H+
NADH H+
e+ H+ H+ ADP ATP
e+ ½ O2 H2O
II
4e+
I I UQ III IV V

Inner cyt c
membrane H+ H+ H+ H+
H+ H+ H+ H+
O
.
OH O
e+ CH3O CH3 e+
CH3O CH3 CH3O CH3

CH3O R CH3O R CH3O R

OH OH O
Ubiquinol Ubisemiquinone radical Ubiquinone
13

III. Reactive oxygen species generation

by mitochondria – chemistry

O O O2 O 2.- H2O 2 HO . H2O

Molecular Superoxide Hydrogen Hydroxyl

Water
2s 2p oxygen anion peroxide radical

O2 O2.-

.
OH O O
CH3O CH3 e+ CH3O CH3 e+ CH3O CH3

CH3O R CH3O R CH3O R

OH OH O
1-3% of electrons are believed to leak from the electron transport chain
to become superoxide
Biochem. J., 1972 Jul., 128(3): 617-30 and Adv. Exp. Med. Biol. 1977, 78: 67-82 14

III. Reactive oxygen species generation

by mitochondria
Succinate
NADH H+ H+
e+ H+ H+ ADP ATP
e+ ½ O2 H2O
II
4e+
I I UQ III IV V
Inner .-
cyt c

membrane H+ H+ H+ H+ H+ H+
O2 O2.-

OH O. O
CH3O CH3 e+ CH3 O CH3 e+ CH3O CH3

CH3O R CH3 O R CH3O R

OH OH O

Steady state levels of reactive components in the respiratory chain

and oxygen (e.g., Ub.-) determine the amount O2.- generated by mitochondria
15

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 Generation of Reactive Oxygen
Species by Mitochondria
Dr. Derick Han
Dr. Enrique Cadenas
III. Sites and mechanism
of reactive oxygen species generation
by mitochondria
Matrix
Succinate
NADH
e+ Antimycin KCN
e+
II

I I UQ III IV V

Inner cyt c
membrane
Rotenone
Mitochondria inhibitors greatly increase superoxide generation
Based on
in the respiratory chainworks
becausewith inhibitors,
they complex
increase steady stateI levels
and III
of reactive
areintermediates
believed to in be
the the
respiratory
majorchain such as
sources of ubisemiquinone
superoxide radical;
Basal levels of reactive
in the oxygen species
mitochondrial generation
respiratory from mitochondria
chain
are difficult to observe without treatment of inhibitors
16

III. Sites and mechanism

of reactive oxygen species generation
by mitochondria: complex I
Matrix

NADH
O2 O.-
2
e+
II

I I UQ III IV V

Inner cyt c
membrane Rotenone

Rotenone greatly increases superoxide generation from complex I;

A flavoprotein component of complex I is believed to be responsible
for superoxide generation, however this component
has not been clearly established; Rotenone induces complex I generation
of superoxide only towards the matrix
Molecular Pharmacology (2003) 64: 1136-1144 17

III. Sites and mechanism

of reactive oxygen species generation
by mitochondria: complex II
Succinate TTFA
Matrix e+

II

I I UQ III IV V

Inner cyt c
membrane

Inhibition of complex II using TTFA has not been shown to cause increase
of reactive oxygen species from mitochondria; Complex II is not believed
to be a source of superoxide in the electron transport chain

Molecular Pharmacology (2003) 64: 1136-1144 18

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 Generation of Reactive Oxygen
Species by Mitochondria
Dr. Derick Han
Dr. Enrique Cadenas
III. Sites and mechanism
of reactive oxygen species generation
by mitochondria: complex III
Matrix
Succinate
NADH
e+ .-
O2 O2 Antimycin
e +
II
UQI - .
II IV V
UQ bL /bH
UQO- . 2Fe/S
Inner cyt c
membrane .-
O2 O2
The ubisemiquinone radical is formed at two sites in complex III –
near the matrix (UQI.- ) and near the intermembrane space (UQO.- );
Antimycin blocks complex III and causes superoxide generation
both towards the intermembrane space and the matrix

Biochem. J., 2001, 353: 411-6; Methods Enzymol., 2002, 349: 271-80
and Arch. Biochem. Biophys., 1985, 237(2): 408-14 19

III. Sites and mechanism

of reactive oxygen species generation
by mitochondria: ubisemiquinone

.-
O2 O2

. O
OH O
CH3 O CH3 e+ CH3O CH3 e+ CH3O CH3

CH3O R CH3O R CH3 O R

OH OH O

The reaction between ubisemiquinone and oxygen

is a thermodynamically favorable reaction
[E(Q/Q.- ) = -220 + 20 mV and E(O2/O2.- ) = -156 mV]

20

III. Sites and mechanism

of reactive oxygen species generation
by mitochondria: complex IV
Matrix
Succinate
NADH
e+
KCN
e+
II
UQI - .
I I UQ IV V
III
UQO- .
Inner cyt c
membrane

KCN and other complex IV inhibitors also increase superoxide generation

by the respiratory chain, likely through increasing steady-state levels
of ubisemiquinone in complex III

21

The screen versions of these slides have full details of copyright and acknowledgements 7
 Generation of Reactive Oxygen
Species by Mitochondria
Dr. Derick Han
Dr. Enrique Cadenas
III. Topology of reactive oxygen species
generation by mitochondria:
antioxidant defenses – superoxide dismutase

O2.- + O2.- + 2H+ H202 + O2 Spontaneous nonenzymatic dismutation

105 M -1S-1
O2.- + O2.- + 2H+ H202 + O2 Enzymatic dismutation
SOD 109 M -1S-1

All superoxide dismutases are metalloproteins containing Cu, Zn, or Mn;

There are three types of superoxide dismutases in humans:

• Cu, Zn-superoxide dismutase Cytosol, (mitochondrial intermembrane space?)

• Mn-s uperox ide dism utase Mitochondrial matrix
• Cu, Zn-superoxide dismutase Extracellular space

22

III. Topology of reactive oxygen species

generation by mitochondria:
antioxidant defenses – mitochondrial GSH
GSH is the major antioxidant
in mitochondria; Mitochondria
do not make GSH, it is imported
into mitochondria

Responsible for detoxification

of hydrogen peroxide generated
by the respiratory chain in mitochondria

H2O2 + 2GSH → H2O + GSSG – catalyzed

by GSH peroxidase

GSSG is reduced back to GSH

by GSSG reductase, which uses
the reducing power of NADPH

Annu. Rev. Pharmacol. Toxicol., 1985, 25: 715-44 23

III. Superoxide generated towards the matrix

NADP+ GSH reductase NADPH
Matrix
GSH GSSG
GSH
Mn-SOD peroxidase

O2 O2.- H2O2 H2O

NADH
O2.-
Inner e+ O2
membrane II
UQI - .
I I UQ III IV V
UQO- .
Inter membrane cyt c
space

Outer membrane H2O2 VDAC

Superoxide generated towards the matrix by complex I and III is converted to H2 O2

by Mn-SOD that resides in the matrix; H2O2 is subsequently reduced to water
by GSH peroxidase which uses the reducing power of GSH; Since H2O2 is membrane
permeable, a small portion of H2O2 diffuses from the matrix to the cytoplasm 24

The screen versions of these slides have full details of copyright and acknowledgements 8
 Generation of Reactive Oxygen
Species by Mitochondria
Dr. Derick Han
Dr. Enrique Cadenas
III. Superoxide generated
towards the intermembrane space
Matrix
NADP+ GSH reductase NADPH

Mn-SOD GSH GSSG

GSH
NADH peroxidase

Inner e+
membrane II
UQI - .
I I UQ III IV V
UQO- .
cyt c
Inter membrane space O2 O2.- Cu, Zn-SOD ?

O2.-
Outer membrane VDAC

Superoxide generated towards the intermembrane space by complex III diffuses from mitochondria
to the cytoplasm through voltage dependent anion channels (VDAC) in the outer membrane; A Cu,Zn-SOD
has also been reported to be localized in the intermembrane space, however it generally exists
in an inactive form; Superoxide cannot cross membranes J. Biol. Chem. (2003) 278: 5557-5563 25

Control of superoxide anion diffusion

through VDAC
Superoxide signal intensity

100
100

50 50 Plus BSA

0 0
0 0.5
[DIDS] (mM)
Treatment of mitochondria with DIDS,
an anion channel inhibitor, resulted
in a dose-dependent inhibition
of O2 .– release detected by EPR
J. Biol. Chem. (2003) 27: 5557-5563
0 Control
0.05 0.1
1 0.2 0.3
Dextran sulfate (mM)
Polyv alent anions such as dextran sulfate
modulate VDAC closure, particularly
in the presence of a membrane potential
across the phospholipid bilayer;
Albumin was used to generate
a Nernst potential across the outer membrane
26

Voltage-dependent anion channels (VDAC)

control the release of superoxide anion
from mitochondria to cytosol

O2. – O2. –

Outer
VDAC VDAC membrane
O2. –

ANT ∆Ψ Inner
membrane

J. Biol. Chem. (2003) 278: 5557-5563 27

The screen versions of these slides have full details of copyright and acknowledgements 9
 Generation of Reactive Oxygen
Species by Mitochondria
Dr. Derick Han
Dr. Enrique Cadenas
III. Topology of reactive oxygen species
generation by mitochondria
NADP+ GSH reductase NADPH
Matrix
GSH GSSG
GSH
Mn-SOD peroxidase
O2 O2.- H2O2
NADH H2O
e+ O2 O2.-
II
UQI - .
I I UQ III IV V
UQO- .
Inner cyt c
membrane O2 O2.-

Outer
O2.- VDAC
membrane H2O2
Mitochondria are a major source of both H2O2 and superoxide in cytoplasm 28

Literature of topology of superoxide release

by mitochondria
• Respiratory chain-dependent generation of superoxide anion
towards the mitochondrial intermembrane space, Biochemical Journal (2001)
353: 411-416, Han, D., Williams, E., and E. Cadenas

• Mitochondrial superoxide anion production and release into intermembrane space,

Methods in Enzymology (2002) 349: 271-80, Han, D., Antunes, F., Daneri, F.
and E. Cadenas

• Voltage dependent anion channels control the release of superoxide from mitochondria
to the cytosol, Journal of Biological Chemistry (2003) 278: 5557-5563,
Han, D., Antunes, F. Canali, R., Rettori, D. and E. Cadenas

• Effect of glutathione depletion on sites and topology of superoxide and hydrogen

peroxide in mitochondria, Molecular Pharmacology (2003) 64: 1136-1144,
Han, D., Canali, R., Rettori, D., and N. Kaplowitz

• Topology of superoxide production from different sites in the mitochondrial electron

transport chain, Journal of Biological Chemistry (2003) 278: 5557-5563,
St-Pierre, J., Buckingham, J.A., Roebuck S.J., and M.D. Brand

• Complex III releases superoxide to both sides of the inner mitochondrial membrane,
Journal of Biological Chemistry (2004) 279: 49064-73, Muller, F.L., Liu, Y.
and H.V. Remmen
29

Mitochondria –
a major cellular source of oxygen radicals

O2
H2 O2 H2 O2
O2 . –
O2 .– O2 .–
VDAC
O2
Mitochondrial ROS
• Signal transduction
• Development
• Cell proliferation
• Apoptosis

30

The screen versions of these slides have full details of copyright and acknowledgements 10
 Generation of Reactive Oxygen
Species by Mitochondria
Dr. Derick Han
Dr. Enrique Cadenas

Overview of mitochondrial reactive

oxygen species generation

I. Mitochondria structure and topology

II. Bioenergetics

III. Reactive oxygen species generation by mitochondria

a) Sites and mechanism
b) Topology

IV. Methods to measure reactive oxygen species

V. Regulation of mitochondrial
reactive oxygen species generation

31

Methods to measure H2O2 release

from isolated mitochondria and in cells
Assessing the generation of H2O2 in cells is confounded
H2O2 by the lack of a sensitive and specific assay

1) Horseradish peroxidase (HRP) based assays

HRP
– p-hydroxyphenylacetate (pAH) + H2O2 di-pAH + H2O
(non-fluorescent product) (fluorescent product)

Other used dyes used with HRP include Amplex red, scopletin

This method is specific but cannot be used inside cells

2) DCFH and other redox fluorescent dyes

– Can be used in cells
but are not specific for H2O2 H2 O2 H2 O2

32

Use of DCFH to measure H2O2

DCFH does not react with H2O2
but rather is oxidized by more reactive radicals
like hydroxyl radical and peroxynitrite;
DCF fluorescence is affected by antioxidants levels,
oxidases, and other redox reactive compounds;
DCF is therefore not a measure of H2 O2
but a redox indicator

DCF catalytically stimulates the formation of reactive

Intracellular reactive oxygen
species are detected using probes
like DCFH which become
fluorescent upon oxidation to DCF
oxygen species in a manner that is dependent
on and affected by various biochemical reducing agents;
T his study, together with our earlier studies,
demonstrates that DCFH cannot be used conclusively
to measure superoxide or hydrogen peroxide formation
in cells undergoing oxidative stress
Phenoxyl free radical formation during the oxidation
of the fluorescent dye 2',7'-dichlorofluorescei n by horseradish
peroxidase; Possible consequences for oxidative stress
measurements, Rota C, Fann YC, Mason RP,
J. Biol. Chem., 1999 Oct 1, 274(40): 28161-8
33

The screen versions of these slides have full details of copyright and acknowledgements 11
 Generation of Reactive Oxygen
Species by Mitochondria
Dr. Derick Han
Dr. Enrique Cadenas
Methods to measure superoxide release
from isolated mitochondria and in cells
Assessing the generation of O2•− in cells is confounded
Superoxide by the lack of a sensitive and specific assay

1) Hydroethidine and other fluorescent

dyes (lucigenin, luminol)
Many O2 • − sensors react with intracellular oxidoreductases and can artificially
generate a “superoxide” signal [e.g., hydroethidine (HE), nitroblue tetrazolium,
epinephrine, lucigenin, and luminol]

2) Electron spin resonance (EPR)

+ spin traps
- Need v ery specialized equipment

3) Cytochrome c or acetyl cytochrome c

reduction O2
- Can be used only in isolated mitochondria
O2.–
34

Measurement of superoxide using hydroethidine

Hydroethidine

Etd fluorescence is affecting, by binding to DNA,

membrane potential; In addition, HE may also be oxidized by other
intracellular processes, involving oxidases or cytochromes,
to yield Etd+; Consequently, increased Etd+ fluorescence
is not necessarily proof of O 2•− production; In 2003, Zhao et al.
reported that HE is oxidized by O 2•− to yield a hydroxylated
MitoSox product (HO-Etd+); However, detection of HO-Etd+
by fluorescence microscopy is confounded, because its emission
spectrum strongly overlaps the emission of Etd+

Selective fluorescent imaging of superoxide in vivo using ethidium-based probes

Kristine M. Robi nson *, Micha el S. Janes* †, Marian a Pehar ‡, Jeffrey S. Monette*, Meredith F. Ross § ,
Tory M. Hagen*, Mich ael P. Murp hy § ,and Joseph S. Beckma n*¶
Proc. Natl. Acad. Sci. USA, 2006 October 10, 103(41): 15038–15043 35

Overview of mitochondrial reactive

oxygen species generation

I. Mitochondria structure and topology

II. Bioenergetics

III. Reactive oxygen species generation by mitochondria

a) Sites and mechanism
b) Topology

IV. Methods to measure reactive oxygen species

V. Regulation of mitochondrial
reactive oxygen species generation

36

The screen versions of these slides have full details of copyright and acknowledgements 12
 Generation of Reactive Oxygen
Species by Mitochondria
Dr. Derick Han
Dr. Enrique Cadenas

V. Regulation of mitochondrial reactive

oxygen species generation

a. Rate of respiration by the electron transport chain –

inhibitors, uncoupler protein

b. Mitochondrial GSH depletion

c. Ceramide

d. Nitric oxide

e. Post-translational modification

37

a) Rate
a) Rate
of respiration
of respiration
by thebyelectron
the electron
transport
transport
transport
chain chain
–chain
state––3uncoupled
inhibition
and 4
Experimental condition

State Substrate ADP O2 H2 O2

lev els consumption generation

State 4 High Low Moderate Moderate

(~ 0.2 nmol/min/mg)

State 3 High High High Very low

(~ 0.02 nmol/min/mg)

Uncoupled High Not High Very low

relevant (?)

Inhibited High Not Very low High

relevant (~ 2 nmol/min/mg)

Uncoupled High Not Very low Very high

& inhibited relevant (~ 3.5 nmol/min/mg)

Biochem. J. 1980 Apr 15, 188(1): 31-7; Molecular Pharmacology (2003) 64: 1136-1144
and Physiol. Rev. 1979 Jul, 59(3): 527-605 38

Uncoupler protein (UCP) as a regulator

of mitochondrial ROS generation
A role for uncoupling protein-2 as a regulator of mitochondrial
hydrogen peroxide generation
Nègre-Salv ayre A, Hirtz C, Carrera G, Cazenave R, Troly M, Salv ayre R, Pénicaud L,
Casteilla L
FASEB J., 1997 Aug, 11(10): 809-15
Altogether, these results strongly suggest that UCP2 is sensitive to GDP and that the UCPs,
particularly UCP2, are able to modulate H2 O2 mitochondrial generation; This supports a role
for UCP2 in cellular (patho-) physiological processes involving free radicals generated
by mitochondria, such as oxidative damage, inflammation, or apoptosis

Respiratory uncoupling by UCP1 and UCP2 and superoxide generation

in endothelial cell mitochondria
Fink BD, Reszka KJ, Herlein JA, Mathahs MM, Siv itz WI
Am. J. Physiol. Endocrinol. Metab., 2005 Jan, 288(1): E71-9, Epub 2004 Aug 31
UCP1 and UCP2 overexpression both increased the proton conductance
of endothelial cell mitochondria, as rigorously determined by the kinetic relationship
of respiration to inner membrane potential; However, despite uncoupling,
neither UCP1 nor UCP2 altered superoxide formation
39

The screen versions of these slides have full details of copyright and acknowledgements 13
 Generation of Reactive Oxygen
Species by Mitochondria
Dr. Derick Han
Dr. Enrique Cadenas
a) Rate of respiration by the electron transport
chain – inhibition and uncoupling
Experimental condition

State Substrate ADP O2 H2 O2

lev els consumption generation

State 4 High Low Moderate Moderate

(~ 0.2 nmol/min/mg)

State 3 High High High Very low

(~ 0.02 nmol/min/mg)

Uncoupled High Not High Very low

relevant (?)

Inhibited High Not Very low High

relevant (~ 2 nmol/min/mg)

Uncoupled High Not Very low Very high

& inhibited relevant (~ 3.5 nmol/min/mg)

Biochem J., 1980 Apr 15, 188(1): 31-7; Molecular Pharmacology (2003) 64: 1136-1144
and Physiol. Rev. 1979 Jul, 59(3): 527-605 40

a) Rate of respiration by the electron transport

chain – reverse electron flow
Matrix Succinate

O2.- O2
Mn-SOD e+
H2O2

e+ II
Inner
membrane I I e+ UQ III IV

cyt c

Rotenone
Succinate treatment alone will cause a high rate of H2 O2 generation in mitochondria
isolated from certain organs (e.g., heart, brain), through a process called rev erse
electron flow; Electrons from succinate flow back to complex I and generate
high lev els of superoxide and H2 O2 (~ 2 nmol/min/mg for heart mitochondria);
This process can be inhibited by rotenone treatment; Whether this process
is physiological remains in question
Molecular Pharmacology (2003) 64: 1136-1144 41

V. Regulation of mitochondrial reactive

oxygen species generation

a. Movement of the electron transport chain –

inhibitors, uncoupler protein

b. Mitochondrial GSH depletion

c. Ceramide

d. Nitric oxide

e. Post-translational modification

42

The screen versions of these slides have full details of copyright and acknowledgements 14
 Generation of Reactive Oxygen
Species by Mitochondria
Dr. Derick Han
Dr. Enrique Cadenas

Mitochondrial GSH
GSH is the major antioxidant
in mitochondria; Mitochondria
do not make GSH, it is imported
into mitochondria

Responsible for detoxification

of hydrogen peroxide generated
by the respiratory chain in mitochondria

H2O2 + 2GSH → H2O + GSSG – catalyzed

by GSH peroxidase

GSSG is reduced back to GSH

by GSSG reductase, which uses
the reducing power of NADPH

Mitochondrial GSH levels (mM)

are over a 1000 fold greater
than H2O2 (nM-uM) levels in the matrix
43

Effect of GSH depletion

on H2O2 production from complex I
GSH depleted (95% mitochondria)
Fluorescence (lnt)

Rotenone Severe GSH depletion

increases H2O2 generation
from mitochondria several folds

Control
Glu/mal mitochondria
Rotenone
Han, D., Canali, R., Rettori, D.
and N. Kaplowitz, Effect of glutathione
depletion on sites and topology of superoxide
and hydrogen peroxide in mitochondria,
Molecular Pharmacology (2003)
64: 1136-1144
Time (min)
Glu/mal -glutamate/malate (complex I substrates), rotenone (complex I inhibitor);
H2O 2 was measured using horseradish peroxidase and p-hydroxyphenylacetate (pAH) 44

Effect of GSH depletion on H2O2 production

Hydrogen peroxide (% increase)

Mitochondria
were treated
with glutamate/malate
plus rotenone

GSH (% depleted)

The release of H2O 2 from mitochondria depends on the extent of GSH depleted
and the30-40%
About rate of superoxide generation
of mitochondrial GSH must be depleted
There are abefore increase associated
few pathologies in H2 O2 release from mitochondria
with mitochondrial can be observed
GSH depletion;
Chronic alcohol intake is associated with selective mitochondrial GSH depletion (~ 40-50%) in liver

Molecular Pharmacology (2003) 64: 1136-1144 45

The screen versions of these slides have full details of copyright and acknowledgements 15
 Generation of Reactive Oxygen
Species by Mitochondria
Dr. Derick Han
Dr. Enrique Cadenas

V. Regulation of mitochondrial reactive

oxygen species generation

a. Movement of the electron transport

chain – inhibitors, uncoupler protein

b. Mitochondrial GSH depletion

c. Ceramide

d. Nitric oxide

e. Post-translational modification

46

Ceramides

Sphingomyelin
Sphingomyelinase
Ceramide

Ceramide

Ceramides are released from membranes through the action

of sphingomyelinase or phospholipase C; Once released, ceramides
can modulate many signaling pathways; Tumor necrosis factor (TNF)
is believed to use ceramide as a second messenger; Several reports suggest
that ceramide can translocate to mitochondria and modulate respiration
and reactive oxygen species generation
47

Regulation of mitochondrial
reactive oxygen species generation by ceramide
Ceramide increases ROS generation in isolated mitochondria

Figure 3. Structural specificity and dose-dependent effect of ceramide in the generation of hydrogen peroxide
from isolated mitochondria; A, freshly isolated mitochondria (1 mg/ml) were incubated with 1 µM C2-ceramide, C6-ceramide,
or dihydro-C2 (C2DH) for 60 min in the presence of DFCDA (2 µM); Sphingosine (Sphs ), sphinganine (Sphn),
and sphingomyelin (Sphm) were present at same concentration as ceramides; Increasing their concentration to 5 µM
did not modify fluorescence of DCF; After 60 min of incubation, an aliquot was transferred to the spectrofluorometer to determine
fluorescence of DCF as described under "Materials and Methods“; Control incubation was performed in the presence of vehicle
Me2SO, the solvent used in the stock solution of sphingolipids; Mean fluorescence was correlated with hydrogen peroxide using
known concentrations of the latter (27); B, isolated mitochondria (1 mg/ml) were incubated with increasing concentrations of C2-
ceramide (open circles ) and C2DH (closed circles ) for 60 min and hydrogen peroxide was determined from DCF fluorescence
in a spectrofluorometer as described under "Materials and Methods“; The bar represents the fluorescence of control
mitochondria incubated with Me2SO (0.5 %); Results are mean ± S.D. of three separate observations. *, p < 0.05 versus control

Direct effect of ceramide on the mitochondrial elec tron transport chain leads to generation of reactiv e oxygen species.
Role of mitochondrial glutathione, García-Ruiz C, Colell A, Marí M, Morales A, Fernández-Checa JC,
J. Biol. Chem. 1997 Apr 25, 272(17): 11369-77
48

The screen versions of these slides have full details of copyright and acknowledgements 16
 Generation of Reactive Oxygen
Species by Mitochondria
Dr. Derick Han
Dr. Enrique Cadenas

V. Regulation of mitochondrial reactive

oxygen species generation

a. Movement of the electron transport

chain - inhibitors, uncoupler protein

b. Mitochondrial GSH depletion

c. Ceramide

d. Nitric oxide

e. Post-translational modification

49

Nitric oxide – a physiological regulator

of mitochondrial function
.
Sites of action of NO on the mitochondrial respirator y chain

FAD
II ↓
III IV
[Fe–S]6
I ↓ Antimycin A CN– , CO

bT bK c1 c
NADH FMN  [Fe–S]6 UQ aa3 O2

O2
NO NO NO
O2 .–

50

Inhibition of state 3 active mitochondrial

respiration by nitric oxide
of mitochondrial respiration (% )

The reversible inhibition of cytochrome

100 oxidase by . NO exerts a regulatory control



on mitochondrial respiration

Mitochondria on state 3 respiration show

Inhibition

a higher sensitivity to inhibition by .NO

50
Mitochondrial respiration is controlled
by the availability of ADP to F 1-ATPase

and of O 2 and . NO to cytochrome oxidase

ADP NO O2
0
150 300

[O2 ]/[NO]
cytochrome
F 1–ATPase
Cleeter et al. (1994) FEBS Lett. 345: 50 oxidase
Brown, G.C. (1995) FEBS Lett. 369: 136
Torres et al. (1995) Biochem. J. 312: 169
Boveris et al. (1999) Meth. Enzymol. 301: 188 51

The screen versions of these slides have full details of copyright and acknowledgements 17
 Generation of Reactive Oxygen
Species by Mitochondria
Dr. Derick Han
Dr. Enrique Cadenas
Inhibition of cytochrome oxidase
by nitric oxide
NO O2
2+ + k NOon 2+ + 3+ + Model 1
Fea3 –CuB–NO Fea3 –CuB k O Fea3 –CuB–O2
k NO 2 app
off
k IV Model 2

cyt c3+ cyt c2+

• The inhibition of cytochrome oxidase k III
by . NO can be accounted for by a direct
competition between . NO and O 2 • Cytochrome oxidase activity found
for the two-electron reduced bi-nuclear center in tissues is optimized or in excess
Cu B/a3 of cytochrome oxidase to avoid an excessive inhibition
of mitochondrial respiration by .NO
• Cytochrome oxidase is inhibited rapidly,
in a time scale of seconds; half-inhibition • The excess capacity of cytochrome
of respiration attained at O 2/ . NO ratios oxidase allows a wide cellular range of .NO
in the 40-500 range activities without causing excessive
inhibition of the respiratory chain
• The interaction of cytochrome oxidase
with . NO is stronger than that with O 2, despite • Activation of mtNOS results
that the binding of O 2 and .NO to the reduced in an intramitochondrial [.NO]ss facing
binuclear center (Cu B/a3) is similar: ~108 M–1s–1 cytochrome oxidase in the range 100-300 nM

Antunes et al. (2004) Proc. Natl. Acad. Sci. USA 52

Peroxynitrite, a strong oxidant, is formed

from the reaction between . NO and superoxide

NOS

O2.– + .NO ONOO–

1.9 x 1010 M –1s–1

Respiratory
chain

53

Biphasic effect of . NO
on mitochondrial H2O2 production

2 0.14
H2 O2 20
+d[ONOO– ]/dt (10–6 M/s) (
+d[H2 O2 ]/dt (nmol/min/mg)

ONOO–
[O2 – ]ss (10–10 M) ( )

● ●●
●

●
1 0.07 .
[O2 – ]ss ● 10

●
.

●
)

0 0
0 5
. 10 15 20
[ NO] (µM)

O2.– + .NO ONOO–

1.9 x 1010 M –1s–1
Arch. Biochem. Biophys., 1996 Apr 1, 328(1): 85-92 54

The screen versions of these slides have full details of copyright and acknowledgements 18
 Generation of Reactive Oxygen
Species by Mitochondria
Dr. Derick Han
Dr. Enrique Cadenas

Mitochondrial generation of reactive oxygen

and nitrogen species
. NO

NADH
UQH.
DH
.– mtNOS
O2
. NO
H2 O2
VDAC

H2 O2 O2.
– . NO

ONOO-

55

V. Regulation of mitochondrial reactive

oxygen species generation

a. Movement of the electron transport

chain – inhibitors, uncoupler protein

b. Mitochondrial GSH depletion

c. Ceramide

d. Nitric oxide

e. Post-translational modification

56

Post translational protein modifications

Disulfide linkage
H2O2
Tyrosine nitration S S
ONOO–

NO2–Tyr
NO SNO
ONOO–
SH S-nitrosylation GSH
Tyr

PO4 2– S–SG
GSSG

S-glutathionylation

SOH
O–PO3– H2O2
Phosphorylation ONOO–
Sulfenic acid

Am. J. Physiol. Gastrointest. Liver Physiol. 2006 Jul, 291(1): G1-7

57

The screen versions of these slides have full details of copyright and acknowledgements 19
 Generation of Reactive Oxygen
Species by Mitochondria
Dr. Derick Han
Dr. Enrique Cadenas
Mitochondrial protein
post-translational modifications
S-Glutathionylation
S-Nitrosylation
Energy metabolism
Energy metabolism
aconitase
citrate synthase
succinyl-CoA transferase
glutamate dehydrogenase
pyruvate dehydrogenase
Electron transfer
succinyl CoA synthase
complex V
complex I Electron transfer
complex I
Nitration complex IV
Energy Metabolism complex V
aconitase Prohibitin
succinyl-CoA transferase Apoptosis
VDAC - ANT
Phosphorylation creatine kinase
Energy metabolism adenylate kinase 2
pyruvate dehydrogenase
Transport
H+/phosphate symporter
Electron transfer
oxoglutarate/GSH carrier
complex IV

Apoptosis
Bcl-2, Bcl-xL
58

S-glutathionylation of aconitase
decreases activity
Protein–SH + GSSG → Protein–SSG + GSH
Aconitase activ ity

0 25 50 100 200
[GSSG] (µM)
Aconitase thiols

Glutathionylated
aconitase

0 0.1 0.2 0.5 1.0 0 0.1 0.2 0.5 1.0

[GSSG] (mM) [GSSG] (mM)
Biochemistry, 2005 Sep 13, 44(36): 11986-96 59

Post translational modification

of complex I increases ROS generation

S-nitrosothiol inhibition of mitochondrial complex I causes

a reversible increase in mitochondrial hydrogen peroxide production

Vilmante Borutaite and Guy C. Brown

Biochimica et Biophysica Acta (BBA) - Bioenergetics
Volume 1757, Issues 5-6, May-June 2006, Pages 562-566

Reversible glutathionylat ion of complex I increases mitochondrial

superoxide formation

Taylor ER, Hurrell F, Shannon RJ, Lin TK, Hirst J, Murphy MP

J. Biol. Chem. 2003 May 30, 278(22): 19603-10

60

The screen versions of these slides have full details of copyright and acknowledgements 20
 Generation of Reactive Oxygen
Species by Mitochondria
Dr. Derick Han
Dr. Enrique Cadenas

Modifying the cell’s redox status

Oxygen O2 RNH2
Mitochondrial Nitric oxide
+1e– synthase –5e–
ubiquinol
Superoxide .
anion O2 – . Nitric
NO
+1e– ONOO– oxide
Hydrogen Peroxynitrite
peroxide H2O2

Univalent oxygen reduction

and nitric generation yield the major
reactive oxygen- and nitrogen species

61

Post translational modification of proteins

by reactive oxygen and nitrogen species

Oxygen Nitrogen
metabolism metabolism

Oxidative stress Nitrosative/Nitrative

stress

Chemical modifications of target proteins

Cysteine
Irreversible Reversible disulfide linkage
modifications modifications S-nitrosylation
S-glutathionylation
oxidation
Tyrosine
nitration
Serine, threonine

62
phosphorylation

Mitochondrial generation of signaling molecules

and regulation of MAPKs
by oxidative and nitrosative stress
. NO

NADH
UQH.
DH

O2
–. mtNOS
. NO
H2 O2
VDAC

.– . NO
H2 O2 O2

ONOO-

Dissociation of Trx-ASK-1 complex S-Nitrosylation

Dissociation of GT-JNK complex ↑ cGMP-dependent kinases
↓ MAPK phosphatase ↓ MAPK phosphatase-3
63

The screen versions of these slides have full details of copyright and acknowledgements 21
 Generation of Reactive Oxygen
Species by Mitochondria
Dr. Derick Han
Dr. Enrique Cadenas

Summary

Mitochondria are major cellular sources of reactive oxygen

species, mainly superoxide anion (O2.–) and H 2O2;
Mitochondria may also be important sources
of peroxynitrite when nitric oxide levels are high

Reactive oxygen and nitrogen species generated

by mitochondria may be important regulators
of redox-sensitive signaling pathways

Consequently reactive oxygen and nitrogen species

generated by mitochondria have important physiological
and pathophysiological roles in cells

64

Acknowledgements

University of Southern California University of Lisbon

Allen Chang Fernando Antunes
Jerome Garcia
Ryan Hamilton
Juliana Hwang
Philip Lam
Lulu Tang
LiPeng Yap
Fei Y in
Qiongqiong Zhou

65

66

The screen versions of these slides have full details of copyright and acknowledgements 22